Association of Preoperative and Postoperative Plasma Syndecan-1 and Colorectal Cancer Outcome.

BACKGROUND/AIM: A three-dimensional network constructed using glycocalyx (GCX) extends throughout the cancer cell nest in human colorectal cancer (CRC). GCX was found to be closely related to cancer. We examined the prognostic correlation and potential of syndecan-1 (SDC1), a representative proteoglycan of GCX, as a biomarker. PATIENTS AND METHODS: We analyzed SDC1 in the transcriptomic profiles of a major publicly available CRC cohort from The Cancer Genome Atlas (TCGA) using a computational algorithm. We investigated serum SDC1 levels preoperatively and on postoperative day seven in 48 patients with stage I-III CRC who underwent surgery during July-December 2019 at Gifu University Hospital. RESULTS: For TCGA, no significant differences existed between the high and low SDC1 expression groups regarding disease-free, disease-specific, and overall survival for stage I-III, and only overall survival for stage IV was significantly different. In our study, among the 48 patients, 17 (no recurrence), 13 (1 recurrence), and 18 (10 recurrences) had stage I-III, respectively. Preoperative and postoperative day 7 SDC1 levels for patients with stage I-III were 10.7±2.3 and 9.9±3.1 ng/ml (p=0.40), 11.1±1.7 and 10.1±0.8 ng/ml (p=0.07), and 10.3±2.0 and 9.5±1.4 ng/ml (p=0.15), respectively. In stage II and III, patients were divided into two groups according to differences between preoperative and postoperative SDC1 levels (SDC1pre-pro). SDC1pre-pro ≤0 group significantly prolonged disease-free survival compared with SDC1pre-pro >0 group (p=0.048). CONCLUSION: Dynamic change in serum SDC1 levels serves as a prognostic biomarker for stage II and III colorectal cancer.

Application of an artificial intelligence-based system in the diagnosis of breast ultrasound images obtained using a smartphone.

BACKGROUND: Breast ultrasound (US) is useful for dense breasts, and the introduction of artificial intelligence (AI)-assisted diagnoses of breast US images should be considered. However, the implementation of AI-based technologies in clinical practice is problematic because of the costs of introducing such approaches to hospital information systems (HISs) and the security risk of connecting HIS to the Internet to access AI services. To solve these problems, we developed a system that applies AI to the analysis of breast US images captured using a smartphone. METHODS: Training data were prepared using 115 images of benign lesions and 201 images of malignant lesions acquired at the Division of Breast Surgery, Gifu University Hospital. YOLOv3 (object detection models) was used to detect lesions on US images. A graphical user interface (GUI) was developed to predict an AI server. A smartphone application was also developed for capturing US images displayed on the HIS monitor with its camera and displaying the prediction results received from the AI server. The sensitivity and specificity of the prediction performed on the AI server and via the smartphone were calculated using 60 images spared from the training. RESULTS: The established AI showed 100% sensitivity and 75% specificity for malignant lesions and took 0.2 s per prediction with the AI sever. Prediction using a smartphone required 2 s per prediction and showed 100% sensitivity and 97.5% specificity for malignant lesions. CONCLUSIONS: Good-quality predictions were obtained using the AI server. Moreover, the quality of the prediction via the smartphone was slightly better than that on the AI server, which can be safely and inexpensively introduced into HISs.

Accelerated glycolysis in tumor microenvironment is associated with worse survival in triple-negative but not consistently with ER+/HER2- breast cancer.

Metabolic reprogramming to sustain immortality is a hallmark of cancer and glycolysis is an important way to attain this. Thus, we investigate the association of glycolysis and associated pathways in the survival of breast cancer. A total of 5,176 breast cancer patients from multiple independent cohorts were analyzed. We determined the glycolytic signaling score by the degree of enrichment by Gene Set Variant Analysis and the median was used to divide each cohort into high vs low score groups. Glycolysis high breast cancer significantly enriched the hallmark cell proliferation-related gene sets (E2F targets, G2M checkpoint, and MYC targets v1 and v2) and was associated with high MKI67 expression. In all cohorts, triple-negative breast cancer (TNBC) was associated with the highest glycolysis score. It was found that in TNBC, glycolysis high breast cancer was associated with worse survival but in ER-positive/HER2-negative breast cancer this was not observed consistently. The glycolysis high TNBC enriched multiple pro-cancerous gene sets and was infiltrated with a low level of B-cells and anti-cancerous immune cells, and significantly associated with a decreased level of cytolytic activity. It was also observed that the glycolysis was higher in the metastatic sites than in the primary breast cancer and the survival was not affected by the metastatic sites. In conclusion, accelerated glycolysis is associated with cancer cell proliferation and worse survival in TNBC.

Detection of high-risk patients resistant to CDK4/6 inhibitors with hormone receptor-positive HER2-negative advanced and metastatic breast cancer in Japan (KBCSG-TR-1316).

BACKGROUND: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) improve the prognosis of hormone receptor-positive HER2-negative advanced/metastatic breast cancer (HR+/HER2- mBC). However, some cancers show resistance to CDK4/6i and have a poor prognosis. The non-luminal disease score (NOLUS) was developed to predict non-luminal disease using immunohistochemical analysis. METHODS: The association between the efficacy of CDK4/6i and NOLUS was investigated by evaluating pathological and clinical data, including real-world progression-free survival (rw-PFS) and overall survival (OS). Real-world data of patients with HR+/HER2- mBC who received CDK4/6i therapy [palbociclib or abemaciclib] as first- or second-line endocrine treatments was obtained. NOLUS was calculated using the formula: NOLUS (0-100) = - 0.45 × estrogen receptor (ER) (%) - 0.28 × progesterone receptor (PR) (%) + 0.27 × Ki67(%) + 73, and the patients were divided into two groups: NOLUS-positive (≥ 51.38) and NOLUS-negative (< 51.38). RESULTS: Of the 300 patients, 28 (9.3%) were NOLUS-positive, and 272 (90.7%) were NOLUS-negative. The expression rates (%) of ER and PgR in NOLUS-positive patients were lower than those in NOLUS-negative patients (p < 0.001). Ki67 expression was higher in NOLUS-positive patients. There were statistically significant differences in prognosis (rw-PFS and OS) between the two groups. Moreover, NOLUS-negative patients showed statistically better rw-PFS with first-line therapy than second-line therapy. However, NOLUS-positive patients showed poor prognoses with both the first and second therapeutic lines, suggesting CDK4/6i inefficacy for NOLUS-positive patients. CONCLUSIONS: The efficacy and prognosis of CDK4/6i significantly differed between the NOLUS-positive and NOLUS-negative patients. This feasible method can predict patients with HR+/HER2- mBC resistant to CDK4/6i and help select a better therapeutic approach to overcome resistance.

Neoadjuvant chemotherapy using nanoparticle albumin-bound paclitaxel plus trastuzumab and pertuzumab followed by epirubicin and cyclophosphamide for operable HER2-positive primary breast cancer: a multicenter phase II clinical trial (PerSeUS-BC04).

BACKGROUND: Nanoparticle albumin-bound paclitaxel (nab-PTX) is a promising antibody partner for anti-human epidermal growth factor receptor 2 (HER2). We performed neoadjuvant chemotherapy (NAC) for HER2-positive breast cancer (BC) using nab-PTX plus trastuzumab (T-mab) and pertuzumab (P-mab), followed by epirubicin and cyclophosphamide (EC). METHODS: In this multicenter phase II clinical trial (January 2019-July 2020), patients with stage I (T1c)-IIIB HER2-positive primary BC were treated with four cycles of nab-PTX plus T-mab and P-mab, followed by four cycles of EC. The primary endpoint was the pathological complete response (pCR) rate. Secondary endpoints were clinical response rate (RR), adverse events (AE), and tumor-infiltrating lymphocytes (TILs) in biopsy samples. RESULTS: In total, 43 patients were enrolled (mean age, 54 years). Twenty-two patients had HER2, and 21 patients had luminal/HER2-subtypes. The overall pCR rate was 53.5% (23/43, 95% CI: 42.6-64.1%, p = 0.184), whilst the pCR for HER2 was 68.2% (15/22, 95% CI: 45.1-86.1) and 38.1% for luminal/HER2 (8/21, 95% CI: 18.1-61.6%). The RR was 100% [clinical (c) CR:25, partial response (PR): 18]. AEs (≥ G3) included neutropenia (23.3%), leukopenia (7.0%), liver dysfunction (7.0%), and peripheral neuropathy (4.7%) when nab-PTX was administered. EC administration resulted in leukopenia (34.2%), neutropenia (31.6%), and febrile neutropenia (15.8%). The TILs in preoperative biopsy samples were significantly higher in pCR compared to non-pCR samples. CONCLUSION: Nab-PTX plus T-mab and P-mab induced a high pCR rate in HER2-positive BC, particularly in the HER2-subtype. Given that AEs are acceptable, this regimen is safe and acceptable as NAC for HER2-positive BC.

Antitumor effects of chemically modified miR-143 lipoplexes in a mouse model of pelvic colorectal cancer via myristoylated alanine-rich C kinase substrate downregulation.

Replenishing tumor-suppressor miRNAs (TS-miRNAs) is a potential next-generation nucleic acid-based therapeutic approach. Establishing an effective miRNA delivery system is essential to successful TS-miRNA therapy. To overcome vulnerability to RNA nucleases, we previously developed a chemically modified miRNA143-3p (CM-miR-143). In clinical practice, colorectal cancer (CRC) pelvic recurrence is an occasional challenge following curative resection, requiring a novel therapy because reoperative surgery poses a significant burden to the patient. Hence, we considered the use of CM-miR-143 as an alternative treatment. In this study, we used a mouse model bearing pelvic CRC adjacent to the rectum and investigated the anticancer effects of CM-miR-143 lipoplexes formulated from miRNA and a cationic liposome. Compared with commercial synthetic miR-143, CM-miR-143 lipoplexes accumulated heavily in regions of the pelvic CRC tumor where the blood flow was high. As a result, systemic administration of CM-miR-143 lipoplexes improved animal survival by significantly suppressing pelvic CRC tumors and relieving a lethal bowel obstruction caused by rectal compression. Detailed protein analysis revealed that the myristoylated alanine-rich C kinase is a novel target for CM-miR-143 lipoplexes. Our results suggest that CM-miR-143 is a potential next-generation drug candidate in the treatment of CRC pelvic recurrence.

Elevated bile acid metabolism and microbiome are associated with suppressed cell proliferation and better survival in breast cancer.

Bile acids are metabolized by the gut microbiome and are involved in fat absorption. Contrary to their carcinogenic role in gastrointestinal cancers, bile acids have been reported to inhibit cancer cell proliferation in breast cancer. The microbiome of breast cancer tissues may also influence cancer proliferation. We hypothesized that bile acid metabolism reflects its accumulation and is associated with certain microbiomes, breast cancer biology, and patient survival. Transcriptomic and clinicopathological information of a total of 6050 patients in three large open primary breast cancer cohorts (GSE96058, METABRIC, TCGA) and 16S rRNA gene sequence microbiome data of breast cancer tissues in TCGA were analyzed by high and low bile acid metabolism scores calculated by gene set variation analysis (GSVA). Breast cancers with high bile acid metabolism had a significantly improved survival across all three cohorts. Metabolic pathways related to the production and regulation of bile acids were consistently enriched in high bile acid metabolism groups across all cohorts. On the other hand, the low bile acid metabolism group was associated with higher Ki67 expression and Nottingham histological grade, as well as enrichment of cell proliferation-related gene sets. Intratumoral heterogeneity, homologous recombination deficiency, mutational load, activation of cancer immunity, and infiltration of anticancer immune cells were also higher in this group. Gammaretrovirus, Hymenobacter, Anaerococcus, and Collimonas were significantly more abundant in the high bile acid metabolism group compared to Lactobacillus, Ruegeria, and Marichromatium in the low metabolism group. Surprisingly, almost all Hallmark cell proliferation-associated gene sets were highly enriched in all three microorganisms that were abundant in the low bile acid metabolism group. In conclusion, microorganisms abundant in the breast tumor microenvironment with low bile acid metabolism are associated with aggressive cancer biology, including increased cell proliferation and poor survival.

The Role of the Microbiome on the Pathogenesis and Treatment of Colorectal Cancer.

The gut microbiome has long been known to play a role in various aspects of health modulation, including the pathogenesis of colorectal cancer (CRC). With immunotherapy recently emerging as a successful treatment in microsatellite instability high (MSI-high) CRC, and with a newly demonstrated involvement of the gut microbiome in the modulation of therapeutic responses, there has been an explosion of research into the mechanisms of microbial effects on CRC. Harnessing and reprogramming the microbiome may allow for the expansion of these successes to broader categories of CRC, the prevention of CRC in high-risk patients, and the enhancement of standard treatments. In this review, we pull together both well-documented phenomena and recent discoveries that pertain to the microbiome and CRC. We explore the microbial mechanisms associated with CRC pathogenesis and progression, recent advancements in CRC systemic therapy, potential options for diagnosis and prevention, as well as directions for future research.

High miR-99b expression is associated with cell proliferation and worse patient outcomes in breast cancer.

Although miR-99b is a known suppressive microRNA (miRNA) in several cancers, its role in breast cancer has not been elucidated. In this study, we examined the clinical relevance of miR-99b expression in breast cancer. We analyzed miRNA and mRNA expression and their relationships with clinical parameters in 1,961 breast cancer samples from two independent large cohorts, the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA). Several algorithms, including gene set enrichment analysis (GSEA) and xCell, have been used to investigate biological functions and the tumor microenvironment. High miR-99b expression significantly enriched the mTORC1 signaling gene set in breast cancer (NES = 1.63, FDR = 0.03, and NES = 1.58, FDR = 0.10, in METABRIC and TCGA, respectively). No other mechanisms, including the epithelial mesenchymal transition, NFκB, and TGF-ß signaling, were consistently enriched in both cohorts. MiR-99b-high breast cancer was associated with high homologous recombination deficiencies, intratumor heterogeneity, and high rates of mutation and neoantigens. In agreement, miR-99b-high breast cancer was associated with increased cell proliferation, correlating with Nottingham histological grade, and significant enrichment of E2F targets, G2/M checkpoint, and mitotic spindle gene sets consistently in both cohorts (P = 0.01, P < 0.001). High miR-99b levels were also associated with low stromal cell fractions in the tumor microenvironment, including adipocytes, keratinocytes, and lymphatic endothelial cells (P < 0.001). However, in both cohorts, miR-99b expression was not associated with significant infiltration of immune cells, except dendritic cells (P = 0.006, 0.020). Finally, in both cohorts, breast cancer with high miR-99b expression was significantly associated with worse disease-free survival (DSS) and overall survival (OS), particularly in estrogen receptor (ER)-positive/human epidermal growth factor (HER)2-negative breast cancer (DSS hazard ratio (HR) 1.29, 95% confidence interval (CI) 1.10-1.51, P < 0.001 in the METABRIC cohort and HR 1.82, 95% CI 1.12-2.98, P = 0.017 in the TCGA cohort). In conclusion, breast cancer with high miR-99b expression was significantly associated with mTORC1 signaling, cell proliferation, and decreased patient survival, particularly in the ER-positive/HER2-negative subtype.

Higher intra-tumoral expression of pro-coagulation genes is a predictor of angiogenesis, epithelial mesenchymal transition and worse patient survival in gastric cancer.

Coagulation regulates angiogenesis in cancer, and is associated with tumor development and metastasis. To date, there have been no studies quantifying the state of intra-tumoral coagulation. We measured intra-tumoral coagulation gene expression using the "Hallmark-COAGULATION" gene set in the MSigDB, performing gene set variation analysis and then assigning a "coagulation score" to quantify gene expression. Clinical, histologic, and genetic data were analyzed in 807 gastric cancer patients from the TCGA_STAD and GSE84437 databases. Tumors with increased expression of pro-coagulation genes were consistently associated with higher AJCC T-categories (p = 0.018), lymph node metastasis (p = 0.036), and stage (p = 0.006) in both cohorts. Patients with high coagulation scores were found to have worse disease-specific survival and overall survival (OS) (p = 0.019 and 0.011, respectively) in TCGA, and worse OS in GSE84437 cohort (p = 0.012). Higher expression of pro-coagulation genes correlated with increased intra-tumoral angiogenesis, as well as increased proportions of lymphatic and microvascular endothelial cells, endothelial cells, and pericytes, calculated by xCell algorithm. High coagulation scores were significantly associated with low tumor mutation burden, but not with intratumor heterogeneity and homologous recombination deficiency. Gastric cancers with high coagulation scores contained higher amounts of M1 macrophages and dendritic cells, and low numbers of Th1 cells (all P<0.001). Genes for epithelial mesenchymal transition (EMT), myogenesis, apical junction, transforming growth factor (TGF)-ß signaling, and angiogenesis were enriched in high coagulation score-gastric cancers (all false discovery rate <0.25). In conclusion, gastric cancers expressing higher levels of pro-coagulation genes demonstrate increased angiogenesis, EMT, TGF-ß signaling and worse patient prognosis.

Low adipocyte hepatocellular carcinoma is associated with aggressive cancer biology and with worse survival.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, and non-alcoholic fatty liver disease is strongly associated with its development. To explore the role of adipocytes in HCC, we investigated intratumoral adipocytes, also known as cancer-associated adipocytes (CAA). Based on our prior breast cancer findings, we hypothesized that low intratumoral adipocytes would be associated with aggressive cancer biology, worse tumor microenvironment (TME), and clinical outcomes. The Cancer Genome Atlas (TCGA) was used and validated by the Gene Expression Omnibus (GEO) cohort. xCell algorithm was used to quantify intratumoral adipocytes and top 90% were defined as adipocyte high (AH) and bottom 10% as adipocyte low (AL). We found that AL-HCC was significantly associated with worse disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS). AL-HCC were higher-grade, had high MKI67 expression, enriched cell proliferation-related gene sets, and had increased altered fraction, aneuploidy, and homologous recombination defects. Also, anti-cancer immune cells, CD8, Th1, and M1 cells, as well as pro-cancer Th2 cells were increased in AL-HCC. Micro-RNAs miR-122 (associated with cholesterol metabolism) and miR-885 (associated with liver pathologies) were significantly increased in the AL TME. In conclusion, we found that AL-HCC has worse patient outcomes and is biologically more aggressive with enhanced cell proliferation. Our findings take initial steps to clarify the role of adipocytes in HCC.

Enhanced immune response outperform aggressive cancer biology and is associated with better survival in triple-negative breast cancer.

Although the value of tumor-infiltrating lymphocytes is well known, the clinical relevance of an increased immune response, specifically in breast cancer, has not been investigated across large cohorts of patients using computational algorithms. Our hypothesis stated that an enhanced immune response is associated with an improvement in outcomes. To quantify the immune response, we utilized the allograft rejection score correlated with cytolytic activity and with all the other Hallmark immune-related gene sets. The score reflected the amount of infiltrating immune cells that correlated with the immune checkpoint molecule expressions, including CD4+ and CD8+ T cells, T helper type 1 (Th1) and type 2 (Th2) cells, M1 macrophages, B cells, and plasmacytoid dendritic cells (pDC). A high score was associated with high levels of intratumor heterogeneity, homologous recombination defects, mutation rate, histological grade, advanced stage, and lymph node metastasis. Breast malignancy with a high score enriched immune-related gene sets and pro-cancer-related gene sets, including epithelial-mesenchymal transition and KRAS pathway, in ER-positive/HER2-negative and triple-negative breast cancer (TNBC) groups. TNBC had the highest score compared to other subtypes, and was associated with better survival. In conclusion, we found that breast cancer with a high immune response is associated with aggressive cancer biology, but with better survival in TNBC.

The mechanism underlying resistance to 5-fluorouracil and its reversal by the inhibition of thymidine phosphorylase in breast cancer cells.

Fluoro-deoxyuridine monophosphate (FdUMP) is an active metabolite of 5-fluorouracil (5-FU) synthesized through two hypothesized pathways: The orotate phosphoribosyl transferase-ribonucleotide reductase (OPRT-RR) pathway and the thymidine phosphorylase-thymidine kinase (TP-TK) pathway. In the present study, the mechanism underlying 5-FU resistance was investigated, focusing on changes in 5-FU metabolism using MCF-7, 5-FU-resistant MCF-7/5-FUR, MDA-MB-231 and 5-FU-resistant MDA-MB-231/5-FUR breast cancer cells. The amount of FdUMP present following treatment with 5-FU was determined by the density of the upper band of thymidylate synthase detected by western blotting, and its changes were investigated. MCF-7/5-FUR cells exhibited 5-FU resistance (36.6-fold), and showed decreased OPRT (-69.3%) and TK (-42.6%) levels. MDA-MB-231/5-FUR cells also exhibited 5-FU resistance (15.8-fold), and showed decreased TP (-79.0%) and increased TK (+184%) levels. MCF-7/5-FUR and MDA-MB-231/5-FUR cells both showed decreased synthesis of FdUMP by 91 and 86%, respectively. In MCF-7 and MCF-7/5-FUR cells, the synthesis of FdUMP was decreased when 5-FU was combined with an RR inhibitor, indicating that FdUMP was synthesized through the OPRT-RR pathway. The synthesis of FdUMP was decreased when 5-FU was combined with a TP inhibitor in MDA-MB-231 cells and combined with an RR inhibitor in MDA-MB-231/5-FUR cells, indicating that the synthesis pathway of FdUMP was changed from the TP-TK pathway to the OPRT-RR pathway on acquiring resistance to 5-FU. Notably, the synthesis of FdUMP was increased and the resistance to 5-FU was reversed in MCF-7/5-FUR cells (half maximal inhibitory concentration (IC50): 219.9 to 0.093 µM) and MDA-MB-231/5-FUR cells (IC50: 157.3 to 31.0 µM) when 5-FU was combined with a TP inhibitor. In conclusion, the metabolism of 5-FU and the mechanism underlying the resistance to 5-FU differed among cell lines, and inhibition of TP reversed resistance to 5-FU, thus suggesting that the combination of 5-FU and a TP inhibitor may be considered a promising cancer therapy.

The clinical relevance of unfolded protein response signaling in breast cancer.

Protein homeostasis regulated by the Endoplasmic Reticulum (ER) is a recognized process involved in cancer progression. ER stress activates the Unfolded Protein Response (UPR) and has been implicated in a variety of cancers. Given the role of the UPR activation in carcinogenesis, we hypothesized that UPR activation could be associated with pathological progression, higher clinical stage, and worse survival in breast cancer. A total of 4,416 breast cancer patients from multiple independent cohorts were analyzed. We defined the UPR pathway score by the degree of enrichment by Gene Set Variant Analysis and median was used to divide high vs. low score groups in each cohort. High UPR breast cancer significantly enriched not only cell proliferation-related but also other pro-cancerous gene sets consistently in both METABIC and GSE96058 cohort. Majority of UPR pathway score high cells in the bulk tumor were tumor cells compared to other cells, including stromal, T-, B-, and myeloid-cells (P<0.001). UPR score was significantly associated with advanced stage, high grade, and triple negative breast cancer (TNBC) (all P<0.001). High UPR breast cancer was associated with worse patient survival in both cohorts (all P<0.001). Among breast cancer subtype, ER-positive/HER2-negative breast cancer with high UPR was significantly associated with worse survival, but neither HER-positive nor TNBC. High UPR ER-positive/HER2-negative breast cancer was infiltrated with high level of Th1 and Th2 cells, M1 macrophage, and plasma cells. On the other hand, they were significantly infiltrated with high level of several types of stromal cells in tumor microenvironment (all P<0.001). Finally, high UPR metastatic breast cancer was also associated with worse patient survival (P=0.041). UPR signaling is associated with cancer aggressiveness, and worse survival, especially ER-positive/HER2-negative breast cancer subtype.

Intratumoral PDGFB gene predominantly expressed in endothelial cells is associated with angiogenesis and lymphangiogenesis, but not with metastasis in breast cancer.

PURPOSE: Platelet-derived growth factor B (PDGFB) is known to play essential roles in angiogenesis and lymphangiogenesis during development, and tumor growth and vessel stabilization in experimental models. However, whether these findings could be translated to breast cancer patients remains unclear. We hypothesized that PDGFB gene expression is associated with angiogenesis, cell proliferation, and clinical outcomes in breast cancer patients. METHODS: A total of 7635 primary breast cancer patients with full transcriptome and clinical data available from 13 independent cohorts were analyzed using in silico approach. The median value was used to divide each cohort into high and low PDGFB expression groups. RESULTS: High PDGFB gene expression was associated with increased expression of angiogenesis-related genes, higher amount of vascular cell infiltrations, and with enrichment of angiogenesis gene set, lymphangiogenesis-related gene expressions, lymphangiogenesis-related cell infiltrations, and enrichmentof lymphangiogenesis gene set in GSE96058 and validated by TCGA cohorts; however, not with lymphatic metastasis. PDGFB expression was neither associated with cell proliferation as assessed by Ki67 expression nor with Nottingham histological grade, or response to neoadjuvant chemotherapy. We found that PDGFB was most extensively expressed by endothelial and perivascular-like cells in the tumor microenvironment, and minimally by cancer cells consistently in two single-cell sequence cohorts. High PDGFB expression enriched TGFß, epithelial-mesenchymal transition, hypoxia, and cancer stem cell-associated pathways. However, no association with distant metastasis was observed. Disease-specific and disease-free survival were worse in the high PDGFB expression group consistently in TCGA and METABRIC cohorts. CONCLUSION: PDGFB is predominantly expressed in endothelial cells and is associated with angiogenesis and lymphangiogenesis, but not with cellular proliferation or metastasis in breast cancer.

Clinical Relevance of Estrogen Reactivity in the Breast Cancer Microenvironment.

Purpose: Estrogen signals play an important role in the phenotype of estrogen receptor-positive breast cancer. However, comprehensive analyses of the effect of responsiveness to estrogen signals on the tumor microenvironment and survival in large cohorts of primary breast cancer patients have been lacking. We aimed to test the hypothesis that estrogen reactivity affects gene expression and immune cell infiltration profiles in the tumor microenvironment and survival. Methods: A total of 3,098 breast cancer cases were analyzed: 1,904 from the Molecular Taxonomy of Breast Cancer (METABRIC) cohort, 1,082 from The Cancer Genome Atlas (TCGA) cohort, and 112 from the Hokkaido University Hospital cohort. We divided the group into estrogen reactivity-high and estrogen reactivity-low groups utilizing the scores of ESTROGEN_RESPONSE_EARLY and ESTROGEN_RESPONSE_LATE in Gene Set Variation Analysis. Results: Breast cancer with high estrogen reactivity was related to Myc targets, metabolism-related signaling, cell stress response, TGF-beta signaling, androgen response, and MTORC1 signaling gene sets in the tumor microenvironment. Low estrogen reactivity was related to immune-related proteins, IL2-STAT5 signaling, IL6-JAK-STAT3 signaling, KRAS signaling, cell cycle-related gene sets, and EMT. In addition, breast cancer with high levels of estrogen reactivity had low immune cytolytic activity and low levels of immunostimulatory cells. It also had low levels of stimulatory and inhibitory factors of the cancer immunity cycle. Patients with high estrogen reactivity were also associated with a better prognosis. Conclusion: We demonstrated the relationship between estrogen reactivity and the profiles of immune cells and gene expression, as well as survival.

NR2F1, a Tumor Dormancy Marker, Is Expressed Predominantly in Cancer-Associated Fibroblasts and Is Associated with Suppressed Breast Cancer Cell Proliferation.

BACKGROUND: Tumor dormancy is a crucial mechanism responsible for the late recurrence of breast cancer. Thus, we investigated the clinical relevance of the expression of NR2F1, a known dormancy biomarker. METHODS: A total of 6758 transcriptomes of bulk tumors from multiple breast cancer patient cohorts and two single-cell sequence cohorts were analyzed. RESULTS: Breast cancer (BC) with high NR2F1 expression enriched TGFß signaling, multiple metastases, and stem cell-related pathways. Cell proliferation-related gene sets were suppressed, and MKi67 expression was lower in high NR2F1 BC. In tumors with high Nottingham grade, NR2F1 expression was found to be lower. There was no consistent relationship between NR2F1 expression and metastasis or survival. Cancer mutation rates, immune responses, and immune cell infiltrations were lower in high NR2F1 tumors, whereas the infiltration of stromal cells including cancer-associated fibroblasts (CAFs) was higher. NR2F1 was predominantly expressed in CAFs, particularly inflammatory CAFs, rather than in cancer cells, consistently in the two single-cell sequence cohorts. CONCLUSIONS: NR2F1 expression in breast cancer is associated with tumor dormancy traits, and it is predominantly expressed in CAFs in the tumor microenvironment.

Abundance of reactive oxygen species (ROS) is associated with tumor aggressiveness, immune response, and worse survival in breast cancer.

PURPOSE: Reactive oxygen species (ROS) are oxygen-containing molecules that have high reactivity and play roles in protection or harm the cancer cells. We aimed to clarify the clinical relevance of ROS in breast cancer (BC) tumor microenvironment (TME). We hypothesized that it is associated with worse BC patient outcomes. METHODS: ROS score was generated by Gene Set Variation Analysis of Hallmark ROS pathway gene set and a total of 6245 BC patients were analyzed. RESULTS: High ROS BC significantly enriched cell proliferation-related gene sets (MYC targets v1 and v2, G2M checkpoint, E2F targets), pro-cancer-related gene sets (DNA repair, unfolded protein response, MTORC1 signaling, PI3K/AKT/MTOR signaling, glycolysis, and oxidative phosphorylation), immune-related gene sets (inflammatory response, allograft rejection, interferon-α and γ responses, complement, and IL6/JAK/STAT3 signaling), and infiltrated immune cells (CD4+ memory and CD8+ T cells, Th1 and Th2, dendritic cells, Tregs, M1 and M2 macrophages) and B cells, as well as elevated cytolytic activity consistently in both METABRIC and GSE96058 cohorts. Cancer cells were the major source of ROS in BC TME of single-cell sequence (GSE75688) cohort. High ROS was associated with intratumor heterogeneity, homologous recombination defects, mutation rates, and neoantigens, and with clinical aggressiveness in AJCC stage, Nottingham grade and Ki67 expression, as well as worse overall survival in both GSE96058 and METABRIC, and with worse disease-specific survival in METABRIC. CONCLUSION: Abundant ROS in BC patients is associated with abundant mutations, aggressive cancer biology, immune response, and worse survival.

Intratumoral density of regulatory T cells is a predictor of host immune response and chemotherapy response in colorectal cancer.

Regulatory T cells (Tregs) are a subset of CD4+ T lymphocytes known to dampen the host immune response against cancer cells. Within the tumor microenvironment, Tregs are potent facilitators of immune tolerance, and a higher proportion of Tregs compared to cytotoxic T cells predicts a worse outcome in most solid tumors. We studied the association between Treg density, and cancer biology and clinical outcome in colorectal cancer (CRC). We used xCell to estimate intratumoral Tregs in total of 898 CRC patients in the Cancer Genome Atlas (TCGA) and GCE39582 cohorts. High-Treg CRCs enriched immune response-related gene sets; inflammatory response, IFN-γ and IFN-α response, IL2/IL6 signaling, and allograft rejection, and had significantly high infiltration of CD8, CD4, M1 and M2 macrophage, and dendritic cells in both cohorts. While high-Treg CRCs enriched multiple pro-cancer signaling pathways compared to low-Treg CRCs, such as Epithelial Mesenchymal Transition, K-ras, Hypoxia, TGF-ß, TNF-α, and angiogenesis, Treg infiltration was surprisingly associated with earlier CRC stage in TCGA. Notably, in two separate cohorts a higher proportion of Tregs predicted an improved response to chemotherapy. In the GSE28702 cohort, metastatic CRCs with more Tregs showed a significantly better response to mFOLFOX6 versus low-Treg CRC metastases (88.9% response vs. 16.7%, P<0.001). In the GSE72970 cohort, high-Treg CRCs were found to have a 68.8% response to FOLFOX/FOLFIRI without bevacizumab, compared to 44% response in the low-Treg CRCs. Additionally, high-Treg CRCs were associated with increased expression of immune checkpoint molecules PD-L1/PD-L2, CTLA4, TIGIT and BTLA, implying susceptibility to immunotherapy. We also found that CRCs with higher proportions of Tregs were associated with lower amounts of three microorganisms in the tumor: Lachnoclostridium, flavivirus, and Ornithobacterium. In conclusion, we show that amount of Treg in the tumor is a predictor of host immune response and chemotherapy response in CRC.

Intratumoral lymphatic endothelial cell infiltration reflecting lymphangiogenesis is counterbalanced by immune responses and better cancer biology in the breast cancer tumor microenvironment.

Lymphangiogenesis, the generation of new lymphatic vessels from existing ones, results from the dynamic interactions of lymphatic endothelial cells and the tumor microenvironment (TME). It is well known that lymphangiogenesis occurs during the initial stage of metastasis in various types of malignant tumors. However, it is currently not used as a biomarker partially because gold standard method to quantify it is labor and cost intensive. We hypothesized that the quantity of intratumoral lymphatic endothelial cells (iLECs) in the TME is an indicator of lymphangiogenesis and a predictor of metastatic potential and overall survival in breast cancer. We analyzed a total of 4145 breast cancer patients from the Cancer Genome Atlas (TCGA) and GSE96058 by quantifying their iLECs using the xCell algorithm, and correlated these scores with patient survival, tumor grade, and cancer stage. We also assessed various pro- and anti-cancer gene sets for each tumor to characterize tumor behavior and aggressiveness. As we expected, high-iLEC breast cancer demonstrated enriched lymphoangiogenesis and angiogenesis gene sets and was associated with increased expressions of related genes. Also enriched were inflammatory response and immune response-related gene sets; IL2/STAT5 pathway, IL6/JAK/STAT3 pathway, TNFα pathway, allograft rejection, and complement as well as cancer stemness related gene sets like Notch signaling, Hedgehog signaling, epithelial mesenchymal transition, and Wnt beta-catenin signaling. Tumors with high-iLEC showed higher proportions of stromal cells and fewer anti-cancer immune cells. On the other hand, iLEC score did not correlate with patient survival or lymph node metastasis. Surprisingly, breast cancers with fewer iLECs demonstrated enriched E2F Targets, G2M Checkpoint, MYC Targets v1, and MTORC1 signaling which are cancer cell proliferation-related gene sets and exhibited an abundance of pro-cancer immune cells. The amount of iLEC correlated inversely with Ki67 expression and histological grade, which is in agreement that low-iLEC breast cancer was associated with enhanced cancer cell proliferation. In conclusion, while iLECs can be used as a surrogate for lymphangiogenesis in breast cancer, low-iLEC tumors also exhibit features which correspond to aggressive tumor biology, which may explain why the amount of iLECs was not associated with patient survival in our cohorts.